Chapter 2. Health-affecting compounds in Brassicaceae. Muhammad Jahangir Hye Kyong Kim Young Hae Choi Robert Verpoorte

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1 Health-afecting compounds in Brassicaceae Chapter 2 Health-affecting compounds in Brassicaceae Muhammad Jahangir Hye Kyong Kim Young Hae Choi Robert Verpoorte Division of Pharmacognosy, ection Metabolomics, Institute of Biology, Leiden University, Leiden, The etherlands Comp. Rev. Food ci. Food af ;

2 Chapter 2 Abstract Brassicaceae vegetables are considered to be a food in many areas all over the world. Brassica species are not only known for their high fat and protein contents for human and animal consumption, but Brassicaceae vegetables are also recognized as a rich source of nutrients such as vitamins (carotenoids, tocopherol, ascorbic acid, folic acid), minerals (Cu, Zn, P, Mg among others), carbohydrates (sucrose and glucose), amino acids (for example, L-alanine, L-aspartic acid, L- glutamic acid, L-glutamine, L-histidine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine), and different groups of phytochemicals such as indole phytoalexins (brassinin, spirobrassinin, brassilexin, camalexin, 1-methoxyspirobrassinin, 1- methoxyspirobrassinol, and methoxyspirobrassinol methyl ether), phenolics (such as feruloyl and isoferuloylcholine, and hydroxybenzoic, neochlorogenic, chlorogenic, caffeic, p-coumaric, ferulic, and sinapic acids, anthocyanins, quercetin and kaempferol), and glucosinolates mainly including glucoiberin, glucoraphanin, glucoalyssin, gluconapin, glucobrassicanapin, glucobrassicin, gluconasturtiin, and neoglucobrassicin. All these phytochemicals contribute to the reported antioxidant, anticarcinogenic, and cardiovascular protective activities of Brassica vegetables. However, not all members of this family are equal from a nutritional viewpoint, since significant qualitative variations in the phytochemical profiles of Brassica species and varieties suggest differences in the health promoting properties among these vegetables. In this review, Brassica phytochemicals with their nutritional value and health promoting activities are discussed to give an overview of the literature for Brassica as a food crop. Keywords: Brassicaceae, Health effects, Metabolites 14

3 Health-afecting compounds in Brassicaceae 1 Introduction The Brassicaceae (Cruciferae) family is composed of 350 genera and about 3500 species, 42 including some crops of great economical importance such as Brassica napus L., B. rapa L., and inapis alba L. 43, 44 These species are used as food, spices, and as a source of vegetable oils. 45 The Brassicaceae vegetables represent a major part of the human diet 4 being consumed by people all over the world and are considered 42, 49, important food crops in China, Japan, India, and European countries. 50 Over the past 3 decades Brassica production has grown steadily becoming an important source of oil and protein of plant origin for animal and human nutrition, respectively. Rapeseed (canola) ranks currently as the third source of vegetable oil (after soy and palm) and the third leading source of oil meal (after soy and cotton). 51 Brassica is an inexpensive though very nutritive source of food, providing nutrients and health-promoting phytochemicals such as phenolic compounds, vitamins, phytic acid, fibre, soluble sugars, 56 glucosinolates, 57 minerals, polyphenols, 58 fat, and carotenoids (Figure 1). 59 There is currently much interest in identifying phytochemicals with useful biological activity in food 60 and any significant finding related to the presence of valuable compounds in Brassica species will be welcomed by the food industry. 51 There is ever-increasing evidence that a higher consumption of Brassica vegetables, for example, broccoli, cabbage, kale, mustard greens, Brussels sprouts, and cauliflower, reduces the risk of several types of cancer. 61, 62 The anticarcinogenic effect of these vegetables has been attributed to decomposition products of glucosinolates, indoles, and iso-thiocyanates, 63 phytoalexins, and other antioxidants. 64, 65 Indole-3- carbinol, a natural component of Brassica vegetables 66 has an interesting anticarcinogenic potential, acting via different metabolomic and hormonal pathways. 65 It reduces the incidence of tumours in reproductive organs 66 and the growth of human breast cancer cells. 67 Overall, to date, the most promising anticarcinogenic dietary compounds have been detected in cruciferous vegetables and further elucidation of their protective mechanisms and the identification of other active constituents may contribute to the development of highly health supporting Brassica varieties

4 Chapter 2 Polysaccharides Glucose, Fructose, ucrose Glycolysis Indole-glucosinolates Benzoyl-glucosinolates Aromatic amino Acid Biosynthesis Tyrosine Phenylalanine Tryptophan Lipids, Phospholipids, Cholesterol, teroids, -Carotene, Retinol, -Tocopherol etc. Pyruvate hikimate pathway Phenylpropanoids biosynthesis Flavones, Iso-flavones, Flavanols, Flavonoids, Anthocyanins, Tannins, Lignins, Phenylpropanoids etc. Indoles Acetyl-CoA Amino Acid Biosynthesis Oxaloacetate Citrate Malate Citric Acid Cycle Cycle Fumarat e uccinate Isocitrate Aliphatic-glucosinolates Figure 1 General biosynthesis pathway for Brassicacae metabolites Extracts of the different species of the Brassicaceae family show antioxidant effects 74 and decrease oxidative damage, 75 while the juice of some Brassica species has been proved to protect human hepatoma cells from the genotoxic effects of carcinogens. 68 However, compounds such as glucosinolates and phytates may also have a negative effect on human and animal health. For example, glucosinolates and glucosinolate byproducts can be toxic and are responsible for the bitter, hot, and pungent flavours of Brassicaceae vegetables. 76 Also, thiocyanates, isothiocyanates, and oxazolidine-2-thiones have been shown to be goitrogenic. 77 As Brassicaceae vegetables can be a good source of minerals, antinutrients such as phytates can decrease their bioavailability. 78 The purpose of this review is to provide an overview of healthaffecting compounds identified in the Brassica genus. 16

5 Health-afecting compounds in Brassicaceae 2 Vitamins Brassica vegetables contain high levels of vitamins 58 including carotenes, tocopherols, 79 vitamin C, and folic acid 80 (Table 1). It is a well-known fact that the first 3 vitamins have the potential to prevent and treat malignant and degenerative diseases. 79 Broccoli (Brassica oleracea) extracts are protective against reactive oxygen species (RO) presumably due to the presence of vitamin C, quercetin, kaempferol, lutein, zeaxanthin, 81 -tocopherol, -tocopherol, and -carotene. 82 Bioavailabilty is a critical feature in the assessment of the role of these compounds in human health. When 200 g of broccoli was consumed by healthy volunteers, significant changes in serum of both men and women were observed for lutein, whereas for -tocopherol a significant change was detected in women only, whereas no changes were observed for - tocopherol, -carotene, and retinol. 83 Carotenoids In some Brassica species, carotenoid content is two-fold higher than in spinach. 84 ixteen carotenoids were identified in B. chinensis, B. parachinensis, and B. pekinensis, out of which lutein and β-carotene were the most abundant. 85, 86 Lutein has also been isolated from extracts of fresh raw kale (Brassica oleracea var. acephala) 87 and high levels of other carotenoids, mainly -carotene, were also detected. 76, 79, 88 Two other vegetables, Brussels sprouts and green cabbage, have been reported to contain significant amounts of trans- -carotene and cis- -carotene. 89 Carotenoids present in dark green leafy vegetables might be involved in the prevention of several diseases related to oxidative stress. 86 Tocopherols The predominant tocopherol in all Brassica vegetables is α- tocopherol with the exception of cauliflower, which predominantly contains γ-tocopherol. 90 The tocopherol content of rapeseed oil consists of 64% -tocopherol, 35% -tocopherol, and less than 1% is the mixture of -tocopherol and plastochromanol Vitamin C High levels of vitamin C have been reported in Chinese cabbage, broccoli, cauliflower and cabbage 92 (Table 1). The content of this vitamin in different cultivars of cabbage (Brassica oleraceae L.) ranges from 12.0 to mg/100 g

6 Chapter 2 Folic acid Raw broccoli, 94 cauliflower 95 and cabbage contain folic acid 96 (Table 1), a scarce and important vitamin which acts as a coenzyme in many single carbon transfer reactions in the synthesis of DA, RA, and protein components. 97 Folic acid reduces the risk of neural tube defects and may be associated with the reduced risk of vascular disease and cancer, 98, 99 while low folate intake has been identified as a main cause of anaemia. 100 Table 1 Variation of vitamins ( g/g) among different Brassicaceae vegetables on fresh weight basis. Ascorbic α-carotene β-carotene -Tocopherol Folate acid Broccoli a 0.3 b 8.9 b 16.2 b d Kale 186 e 0.6 b 48.6 b 19.2 b Cauliflower a g 1.7 b 0.53 e Chinese 253 a 0.1 c 0.8 c 0.81 f cabbage White a 0.02 b 0.8 b 1.7 b cabbage Brussels sprouts 158 c 1.4 c 1.5 c a = 92 ; b = 79 ; c = 101 ; d = 94 ; e = 95 ; f = 97 ; g = Minerals Brassica plants have been found to be rich in many minerals including calcium and iron. 84 Among the green leafy vegetables, B. oleracea L. acephala (kale) is an excellent source of minerals, 76 accumulating high levels of P,, Cl, Ca, Fe, r, and K (Table 2). 103 Broccoli accumulates e to concentrations many times above that found in soil, which may greatly enhance its health-promoting properties. 104 Different Brassica vegetables such as cauliflower, bok choy (B. rapa) stems and leaves, broccoli (B. oleracea v. botrytis), and kale (B. oleracea v. acephala) are reported to have high mineral contents 96 (Table 2). Interestingly, all these Brassica vegetables exhibit excellent calcium bioavailability. 105 Cabbage leaf (B. oleracea var. capitata) also contains potentially useful amounts of copper, zinc, iron, and a number of other essential minerals and trace elements. 106 Brassica can be cultivated under hydroponic conditions that lead to high levels of nutritionally important minerals such as Cr, Fe, Mn, e, and Zn. Owing to reproducible and high concentration of minerals in the 18

7 Health-afecting compounds in Brassicaceae edible plant tissue small quantities of this enriched plant can be processed to make capsules or tablets that supply 100% of the recommended daily intake of these elements, with the advantage of using a natural plant source. 107 However, the bioavailability of some of these minerals might be reduced by the presence of glucosinolates, phytates, and phenolics Table 2 Variation of minerals ( g/g) among different Brassicaceae vegetables on fresh weight basis Broccoli Kale Cauliflower Chinese cabbage White cabbage Brussels sprouts Ca a b + 17 a + 60 b + 60 b + 13 a Fe a + 2 b a b b a Cu a b a b b a Mg a + 40 b + 22 a + 30 b + 20 b + 12 a K a b a b b a Zn a b a b + 1 b a a a + 40 b + 27 a + 20 b + 10 b + 7 a Mn a + 1 b a b + 1 b a a = 108 ; b = 109. Heavy metals (for example, Mo, B, Co, e, Cd, Pb, Cr, i, Hg, and As) and others such as Cu, Zn, Mn, Fe may be found in high concentration in contaminated soils and have toxic effects on plants, animals, and human beings. 110 The use of metal-accumulating plants to remove toxic metals from soil is known as phytoremediation 22 and Brassica species such as B. oleracea and B. napus, known for their metal accumulator properties, are used for this purpose. 111 However, this characteristic, which constitutes an advantage for the former use entails, an important toxicological risk if these fruits and vegetables grown in contaminated soils are ingested Lipids Rapeseed oil is one of the most common edible oils in the world. Its nutritive value is excellent due to its unsaturated fatty acid content. 113 Mustard oil is also a significant source of unsaturated fatty acids 19

8 Chapter 2 containing about 94.2%, and only 5.4% saturated fatty acids. These are recognized as essential dietary elements with important effects on human health. 114 Mustard oil contains linolenic acid 21.4% (omega 3), palmitic acid 2.9%, palmitoleic acid 0.2%, stearic acid 1.0%, oleic acid 19.4%, linoleic acid (omega 6) 9.7%, and erucic acid 44.4% 115, showing an inhibition of mutagenicity. 114 Oil content in seeds of different B. campestris genotypes varies from 38.9% to 44.6% and major fatty acids found are oleic, linoleic, linolenic, eicosaenoic, erucic acid ranging from 10.1% to 17.3%, 5.9% to 14.5%, 5.2% to 15.0%, 7.7% to 13.7% and 39.6% to 59.9%, respectively. 116 Canola seed oil is one of the richer sources of omega-3-unsaturated fatty acids 65 and in particular of α- linolenic acid. 117 The oil of commercial B. napus L. is rich in oleic acid and contains moderate levels of linoleic and linolenic acid. 118 Cauliflower is considered to be a food of high nutritional value and some authors relate its quality to the stability of its fatty acids. Environmental stress may enhance the fatty matter content (linolenic acid) and polyphenols. 119 The essential oil of B. rapa var. perviridis leaves was found to contain 48 volatile components, representing % of the oil. The main constituents were found to be 3-butenyl-isothiocyanate ( %), 4-pentenyl isothiocyanate ( %), 2-methyl 5-hexenenitrile ( %), 2-phenylethyl isothiocyanate ( %), and phytol ( %). 84 Volatile chemicals emitted by rape seed oil also contain monoterpenes (limonene, sabinene, β-myrcene, and cis-3-hexen-1-ol acetate), sesquiterpenes, short-chain aldehydes and ketones, other green leaf volatiles and organic sulfides including the respiratory irritant, dimethyl disulfide. 120 The emission of volatiles from cabbage, consisted mainly of monoterpenes (sabinene, limonene, -thujene, 1,8-cineole, - pinene, myrcene, -pinene, and -terpinene). (Z)-3-Hexenyl acetate, sesquiterpene (E, E)- -farnesene, and homoterpene (E)-4, 8-dimethyl-1, 3, 7-nonatriene were emitted mainly from herbivore-damaged plants. 121 In Brassica oils, triacylglycerols are the main constituents making up about 98% of the oils. The remaining nonglyceridic fraction consists of different lipophylic phytochemicals such as tocopherols, sterols, and sterol esters. 122 imilarly, in Brassica oils the remaining 2% consists of sterols, phospholipids, and sphingolipids. The major sterols were identified as stigmasterol, 123 sitosterol, campesterol, and cholesterol, 122 the phospholipids as phosphatidylethanolamine and phosphatidylcholine, and the sphingolipids as cerebrosides

9 Health-afecting compounds in Brassicaceae For the purpose of human nutrition, a high ingestion of oleic acid, linoleic and linolenic acids is advantageous. 118 All polyunsaturated fatty acids including both linoleic and linolenic acids are essential because they cannot be synthesized by humans. 125 However, the type of fatty acids in the dietary fat is very important, being considered, for example, as one of the detrimental factors in colon cancer development. Fats containing omega 6-polyunsaturated fatty acids were found to enhance chemically induced colon cancer, 115 while omega-3-polyunsaturated fatty acids reduce it. 75, 115 Consumption of diet rich with canola fat may also alter the fatty acid composition of lipids of adipose tissue, muscle, kidney, and liver. 126 A diet high in trans linolenic acid may increase plasma LDL/HDL cholesterol and total cholesterol/hdl-cholesterol ratios. Careful deodorization prevents the formation of trans-α-linolenic acid and may help to improve the diet Carbohydrates The type and concentration of free sugars influence the flavour of Brassica products. 127 Fructose, glucose, and sucrose are the major soluble sugars found in Brassica. 128 A comprehensive evaluation of the nutritive profiles of Brassica seed meals of yellow-seeded types (B. napus, B. rapa, B. juncea, and B. carinata) and conventional brownseeded (canola) type showed that all contain sucrose (7.5% 8.7%), oligosaccharides (2.3% 2.5%), ash (6.9% 7.0%), and non-starch polysaccharides (20.4% 19.7%). 129 Fructose is the major sugar in the different types of Brassica, representing between 48.8 and 56.9% of the total sugar content in broccoli cvs. Marathon and enshi, respectively, 48.7% (cv. Mirandela) and 53.8% (cv. Murciana) in the other cabbages. Glucose is the second major sugar, while sucrose represents a maximum of 20.5% in broccoli cv. hogun and 11.1% in cv. Murciana. 127 Dietary Fibre It is composed of non-starch polysaccharides 130 and is an important constituent in Brassicaceae vegetables, contributing to prevent colon cancer. 131 In white cabbage (B. oleracea var. capitata) dietary fibre represents one-third of the total carbohydrate content, the other two-third being low-molecular weight carbohydrates, including glucose (37%), 132, 133 uronic acid (32%), arabinose (12%), and galactose (8%). The dietary fibre content of 6 cultivars of white cabbage (B. oleracea var capitata) was evaluated finding that of the average total dietary fibre of 21

10 Chapter mg/g of dry matter, approximately 25% was soluble. 132 Dietary fibre content of other species was found to vary between 271 and 352 mg/g for the yellow-seeded B. napus and brown-seeded B. napus, respectively, 134 with intermediate values in other species, such as cauliflower (302 mg/g of D.W.), broccoli (330 mg/g of D.W.), and cabbage (226 mg/g of D.W.) Protein and Free Amino Acids The defatted meal of Brassica oilseeds is a valuable source of protein for the livestock feed industry 135 and may constitute an important protein source for human nutrition thereby increasing the value of Brassica crops. However, the high temperatures and organic solvents used during the oil extraction process cause denaturation of proteins in Brassica meal. 56 Protein and free amino acid content in rapeseed meal have a high nutritive value, but the utilization of rapeseed/canola as a source of food-grade proteins for human consumption is still limited due to the presence of antinutrients such as glucosinolates, phytates, and phenolics. 113, 136, 137 Therefore, it is used only for animal feeding. 138 There is a variation in protein content in different groups of Brassica, such as B. napus seeds have higher protein solubilities than meals from B. rapa seeds. Meals with higher protein solubility values also have higher foaming capacity values. 139 eeds of rape, B. napus, and related cruciferous oilseed crops, such as B. campestris, B. juncea, B. carinata, and B. nigra are rich sources of edible protein and rapeseed/canola meal, the by-product of the oil-extraction process, contains up to 42.7% 50% 129, 140 protein. The rape seed (B. napus) meal contains napin and cruciferin as storage proteins and oleosin as a structural protein associated with oil bodies. 138, 140 The 2 albumins or napins in oilseed rape and turnip rape are potential food allergens. 141 Free amino acids are involved in secondary plant metabolism and in the production of compounds which directly or indirectly play an important role in plant-environment interactions and human health. A total of 17 amino acids were identified (L-alanine, L-arginine, L-asparagine, L-aspartic acid, glycine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-methionine, L- phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L- 5, 125 valine) in B. oleracea var italica. -methylcysteine sulfoxide, a naturally occurring -containing amino acid, is contained at high concentrations in Brassica vegetables such as broccoli and cabbage. Its 22

11 Health-afecting compounds in Brassicaceae cholesterol-lowering effects have been demonstrated in animals, observing a significant decrease of the serum level of LDL-C (14% decrease) following the oral administration of broccoli (B. oleracea L. var. botrytis L.) and cabbage (B. oleracea L. var. capitata L.) Indoles Plants may respond to pathogen attack by producing phytoalexins. 143 Phytoalexins are a group of structurally diverse 144, 145 molecules that are generally non-specific in their antimicrobial activities , 146 A number of phytoalexins have been isolated from crucifers (Figure 2). 147 R '' R 1 R=H; R =; R =H 2 R=H; R =O; R =H 3 R=OCH 3 ; R =; R =H 4 R=H; R =; R =OCH 3 5 R=OCH 3 ; R =O; R =H CH 3 H ' R O R 12 R=H 13 R=OCH 3 CHO CH 3 R H 3 C OCH 3 6 R=H 2 7 R=O CHO CH 3 CH 3 HO H H CHO R CH 3 O H OCH 3 OCH 3 8 R=CH R=(O)CH 3 OR O OCH 3 H 14 R=H 15 R=CH CO 2 CH 3 OCH 3 R 18 R=H 19 R=OCH 3 CH 3 CH 3 H 20 HO 21 CH 3 2 H OCH 3 O R R ' 23 R=H; R =H 24 R=OCH 3 ; R =H 25 R=H; Figure 2 tructures of cruciferous phytoalexins: 1: brassinin, 2: brassitin, 3: 1- methoxybrassinin, 4: 4-methoxybrassinin, 5: 1-methoxybrassitin, 6: 1- methoxybrassenin A, 7: 1-methoxybrassenin B, 8: cyclobrassinin, 9: cyclobrassinin sulfoxide, 10: cyclobrassinone, 11: dehydro-4-methoxycyclobrassinin, 12: spirobrassinin, 13: 1-methoxyspirobrassinin, 14: 1-methoxyspirobrassinol, 15: 1- methoxyspirobrassinol methyl ether, 16: dioxibrassinin, 17: methyl 1-methoxyinodole- 3-carboxylate, 18: brassilexin, 19: sinalexin, 20: brassicanal A, 21: brassicanal B, 22: brassicanal C, 23: camalexin, 24: 6-methoxycamalexin, 25: 1-methylcamalexin

12 Chapter 2 In Brassica indole phytoalexin (camalexin) synthesis is induced as a response to pathogen attack and RO generating abiotic elicitors. 148, 149 These phytoalexins inhibit the growth of human cancer cells and thus may have a potential use as chemopreventive agents. 64 everal indole phytoalexins found in Brassica vegetables, brassinin, spirobrassinin, brassilexin, camalexin, 1-methoxyspirobrassinin, 1- methoxyspirobrassinol, and methoxyspirobrassinol methyl ether, have been found to possess significant antiproliferative activity against various cancer cells, while others, such as cyclobrassinin, spirobrassinin, brassinin also exhibited chemopreventive activity in models of mammary and skin carcinogenesis. 150 Brassicaceae species contain a range of signalling and regulatory compounds known to be involved in general defence mechanisms activated by pathogen and herbivore attacks on plants. 151 These include salicylic acid, ethylene, H 2 O 2, and jasmonic acid (an acid-derived oxylipin) 79 and signal peptides, such as systemin ome of these are bioactive compounds which exhibited anticancer activity in animals when added to experimental diets. 79 In particular, jasmonic acid and its derivatives, which represent the best characterized class of signal compounds, mediating the defence responses to wounding and herbivore attack in Brassicaceae, have been proved to inhibit the proliferation of human prostate cancer cells, while not affecting normal human blood 64, 160 cells. 8 Phenylpropanoids, Flavonoids and Tannins Phenylpropanoids, flavonoids and other minor compounds (Table 3) are considered to be among the health promoting compounds in Brassicaceae species. 161 Plant polyphenols are multifunctional, having diverse biological activities apart from acting as reducing agents. 162 Phenolics also contribute to the bitter, astringent, and unpleasant flavour of rapeseed, though the threshold of this unpleasant flavour is higher for individual phenolic compounds than for the mixture. 113 In spite of this they are considered to be beneficial and harmless components of rapeseed meal. The contribution of Brassica vegetables to health improvement has generally been associated with their antioxidant capacity and, undoubtedly, phenolic compounds are the major antioxidants of Brassica vegetables. 89, 163, 164 Phenolics is a generic term which refers to a large number of compounds that can be classified in groups, namely, phenolic 24

13 Health-afecting compounds in Brassicaceae acids, flavonoids, isoflavonoids, lignans, stilbenes, and complex phenolic 11, 165 polymers. As mentioned above, these antioxidants have proved to be good for human health and also useful as food preservatives. 166 Mustard seeds have a chemopreventive potential and enhance the antioxidant defence system. Their inclusion in the diet may very probably contribute to reducing the risk of cancer incidence in the human population. 167 A rapeseed phenolic extract has shown a stronger antioxidant activity than many artificial antioxidants 168 and exhibited a greater efficiency on a mole-to-mole basis than natural antioxidants such as vitamin C, vitamin E, and β-carotene. 162 Table 3 Variation of phenolics ( g/g) among different Brassicaceae vegetables on fresh weight basis Quercetin Kaempferol Apigenin Lutein Brassica oleracea L var. 137 a 46 a 6.8 c italica (Broccoli) Brassica oleracea L var 39 a 12 a 2 a 1.3 c botrytis L (Cauliflower) Brassica campestris var 390 a 96 a 45 a 0.2 c chinensis (Chinese cabbage) Brassica rapa L. subsp. 102 b 334 b sylvestris Brassica oleracea var capitata 51 a 8 a 1.4 c (white cabbage) a = 92 ; b = 161 ; c = 101. pecies of the Brassicaceae family are generally rich in polyphenols. Brassica rapa 113 and B. oleracea L. var. botrytis contain a high amount of phenolic compounds. 169 Phenolic contents of several species have been reported, such as Chinese cabbage (1189±125µg/g), broccoli (822±89 µg/g), cauliflower (278±15µg/g), and white cabbage 92, 168 (153±21µg/g) on a fresh weight basis. In the case of broccoli, phenylpropanoids such as ferulic, sinapic, caffeic, and protocatechuic acid were reported to be the most abundant and important bioactive compounds. 170, 171 Four phenylpropanoids (caffeic, p-coumaric, ferulic, and sinapic acid) were identified in the water-soluble phenolic fraction of the leaves of oilseed rape (Brassica napus L. var. oleifera) 172 and gallic, protocatechuic, p-hydroxybenzoic, vanillic, syringic, salicylic, p- coumaric, caffeic, ferulic, and sinapic acid were identified in kale (B. oleraceae L. var. acephala DC.)

14 Chapter 2 The main phenolics in rapeseed meal were determined to be sinapic acid which constitutes over 73% of its free phenolic acid content, 51 while apart from sinapic acid, rapeseed oil also contains 174, 175 vinylsyringol. An efficient peroxynitrite scavenger activity has been described for sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid), which has shown to contribute to the cellular defence against this powerful cytotoxic free radical, thus avoiding peroxynitrite-mediated disorders. 176 Besides the typical seed constituent sinapic acid, large amounts of choline esters of other phenolic acids have been detected in Brassicaceae species, for example, feruloyl- and isoferuloylcholine and hydroxybenzoic acid. 177 Brassicaceae plants accumulate glucose esters (1,6-di-O-sinapoylglucose), gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6-tri-O-sinapoylgentiobiose) of phenolic acids and kaempferol conjugates. 178 Flavonoids are one of the most common and widely distributed groups of plant phenolics. Over 5000 different flavonoids have been described to date and they are classified into at least 10 chemical groups. Among them, flavones, flavonols, flavanols, flavanones, anthocyanins, and isoflavones are particularly common in the human diet. 179 As these compounds have interesting biological activities, these are being used in numerous medical treatments, 180, , 183 connected to cancer-prevention and cardiovascular system protection, including inhibition of oxidative 184, 185 damage. At higher doses, however, flavonoids may act as mutagens, pro-oxidants that generate free radicals, and as inhibitors of key enzymes involved in hormone metabolism. 186 Flavones are involved in various interactions with other organisms, microbes as well as insects and other plants. 187 Pharmacological activities have been described for various flavonoids (for example, quercetin, apigenin, catechins) which have shown an antiinflammatory action by inhibiting cycloxygenase-2 and inducible nitric oxide synthase. 188 The flavonols quercetin, kaempferol, and isorhamnetin 53, are among the flavonoid derivatives present in Brassica species. Two main flavonol glycosides, quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside, are present in broccoli florets. Three minor glucosides of quercetin and kaempferol, isoquercitrin, kaempferol 3-Oglucoside, and kaempferol diglucoside, have also been detected. The quercetin and kaempferol glycosides were present in florets at a level of 43 g/g and 94 g/g of dry weight, respectively. 192 Glycosylated 26

15 Health-afecting compounds in Brassicaceae kaempferol derivatives from the external leaves of tronchuda cabbage (B. oleracea L. var. costata DC) have been reported. 193 Total flavonoid content in Chinese cabbage, broccoli, cauliflower, and white cabbage is 944 µg/g, 316 µg/g, 172 µg/g, and 102 µg/g, on a fresh weight basis, respectively. 92 The accumulation of derivatives of flavonols such as quercetin in Camelina sativa; quercetin and kaempferol in Crambe hispanica var. glabrata; quercetin, kaempferol, and isorhamnetin in Brassica napus; kaempferol and isorhamnetin in inapis alba is reported. 190 The constitutive flavonoids of B. napus, isorhamnetin-3-sophoroside-7-glucoside and kaempferol-3,7-diglucoside, are effective deterrents of armyworm. 44 Analysis of B. alba extracts revealed the presence of 3,5,6,7,8-pentahydroxy-4-methoxy flavone in shoots, as well as 2,3,4,5,6-pentahydroxy chalcone and 3,5,6,7,8- pentahydroxy flavone in roots and root exudates. Apigenin was also found in the shoots and roots. 194 Anthocyanins are potent antioxidants and consequently may be chemoprotective. 195 Brassicaceae plants provide a variety of anthocyanins. Cauliflower and red cabbage showed differences in their anthocyanin profiles: cyanidin-3,5-diglucoside was absent in cauliflower, while it was well represented in red cabbage, together with the characteristic anthocyanin of Brassica genus, cyanidin-3-sophoroside-5- glucoside. The p-coumaroyl and feruloyl esterified forms of cyanidin-3- sophoroside-5-glucoside were predominant in cauliflower, while the sinapoyl ester was mostly present in red cabbage. 196 Red pigmentation of red cabbage is caused by anthocyanins. Red cabbage contains more than 15 different anthocyanins which are acylglycosides of cyaniding. 197 Red radish (Raphanus sativus L.) contains significant amounts of anthocyanins of which 12 acylated anthocyanins were isolated and analyzed spectroscopically to determine their structure. ix of these were identified as anthocyanin glycosides with 1 or 2 phenylpropanoids. 198 Total proanthocyanidins content in brocolli was found to be 12 µg/g and 7 µg/g in cauliflower, calculated over fresh weight. 92 Five lignans, 5 neolignans, 2 sesquilignans, and 1 dilignan were identified in a phytotoxic extract of Brassica fruticulosa. 199 These compounds exhibited interesting antimicrobial, antifungal, and/or herbicidal activities that are believed to participate in plant defence mechanisms. 200 These compounds also have cancer-preventive effects. 65 Tannins have an adverse effect on the nutritive value of rapeseed meal proteins or isolated proteins. 201 These compounds suppress the availability of essential amino acids 202 and may form complexes with 27

16 Chapter 2 essential minerals, proteins, and carbohydrates. 203 Tannins have also a profound inhibitory effect on the digestion of carbohydrates and proteins in particular. 204 In Brassicaceae vegetables different amounts of tannins have been reported. 58 Inositol hexa-phosphate (phytic acid) and condensed tannins are reported in B. carinata, 78 both of which play an important role in iron binding. 203 Cabbage and turnip contain various amounts of phytic acid, tannic acid, and/or oxalic acid. Tannic acid was found at mg/g (fresh weight basis) in cabbage. Levels of both tannic acid and phytic acid can be significantly (p < 0.05) reduced by different blanching methods. 205 The total amount of tannins in rapeseed/canola hulls ranged from to mg/g of oil-free hulls. Insoluble tannins predominated in canola/rapeseed hulls and comprised from 70 to 95.8% of total tannins present. The amounts of sodium dodecyl-sulphate-extractable tannins were comparable to those of soluble tannins but constituted only % of insoluble tannins present Glucosinolates ulfur-containing phytochemicals of 2 different types are present in Brassica (Cruciferae) vegetables (cabbage, broccoli, etc.): glucosinolates and -methyl cysteine sulfoxide. Glucosinolates (Figure 3) are thioglucosides containing a cyano group and a sulfate group. 207 Glucosinolates are derived from amino acid biosynthesis (Figure 1) and are important secondary metabolites in Brassicaceae family, involved in plant defence against pests and diseases. 207 For example glucoiberin, glucoraphanin, glucoalyssin, gluconapin, glucobrassicanapin, glucobrassicin, gluconasturtiin, and neoglucobrassicin are health promoting compounds found in broccoli inflorescences (B. oleracea L., var. italica, cv. Marathon). 55 These compounds have both positive and negative nutritional effects, 208 appearing to possess anticarcinogenic properties, but also quite different toxic effects. 10 The effects of specific glucosinolate degradation products on individual organisms vary and are not always known. If used in excessive quantity, many of these compounds can be highly toxic. 209 Glucosinolates and their concentrations vary among the different groups of Brassicaceae (Table 4). 210 In Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolates were found to be sinigrin and glucobrassicin. Brussels sprouts also had significant amounts of gluconapin. 211 The predominant glucosinolates in broccoli are 4- methylsulfinylbutyl glucosinolate (glucoraphanin), butenyl 28

17 Health-afecting compounds in Brassicaceae glucosinolate (gluconapin), and 3-indolylmethyl glucosinolate (glucobrassicin). 211 Cruciferous vegetables of the Brassica genus (for example, Brussels sprouts, cauliflower, and broccoli) contain high levels of an indolylmethyl glucosinolate commonly known as glucobrassicin. 213 A great number of glucosinolates have been identified in B. oleracea var. capitata f. alba, namely glucoiberin, progoitrin, epiprogoitrin, sinigrin, glucrafanin, gluconapoleiferin, glucoalysin, gluconapin, 4- hydroxybrassicin, glucobrassicanapin, glucobrassicin, gluconasturein, methoxyglucobrassicin, and neoglucobrassicin. 49 Figure 3 Basic structure of glucosinolates The major glucosinolates detected in different varieties of B. oleracea were 2-propenyl, 3-methyl-sulphinylpropyl and indol-3-ylmethyl, which accounted for an average of 35, 25, and 29%, respectively of the total glucosinolate content, while in B. rapa, but-3-enyl represented 86% of the total, with pent-4-enyl and 2-phenylethyl as the other major glucosinolates. The average total glucosinolate content of the flower buds was determined to be 2518 μmol 100 g -1 dry wt. in troncha (B. oleracea var. tronchuda) and 4979 μmol 100 g -1 dry wt in nabo (B. rapa), which is much higher than the highest amounts reported for broccoli (B. oleracea var. italica). 214 As in other Brassicaceae seeds and plants, rapeseed contains up to 5% of glucosinolates, which are partially decomposed during rapeseed processing or storage. When plant material is crushed, as in food preparation or chewing, a thioglucosidase mediated autolytic process is initiated, generating indole-3-carbinol, glucose, and thiocyanate. 215 These, together with other important degradation products, such as isothiocyanates, vinyl-oxazolidinethione, and nitriles, contaminate the crude rapeseed oils, impairing their hydrogenation and transesterification and ultimately may be harmful to human consumption

18 Chapter 2 The main glucosinolate breakdown products of Brassica vegetables are the sinigrin breakdown product 1-cyano-2,3- epithiopropane, the gluconapin hydrolysis product 3-butenyl isothiocyanate, the glucobrassicin metabolite ascorbigen and low concentrations of other indole glucosinolate-derived hydrolysis products such as neoascorbigen and 3,3`-di-indolylmethane. 217 Rapeseed meal, a by-product of rapeseed oil production, also contains glucosinolates which together with phytic acid contribute to its anti-nutritional properties. 218, Table 4 Variation of glucosinolate contents ( g/g) among different Brassicaceae vegetables on dry weight basis Cabbage Broccoli Brussels sprouts Cauliflower Kale Glucoiberin a a + 84 c d Glucoraphanin 17 a a c c b Progoitrin a + 68 a c + 40 c Gluconapin a + 37 a c + 74 c + 37 c inigrin a c + 36 c d Glucoalysin c + 45 c Glucoerucin 1206 b Glucobrassicin a a c c c eoglucobrassicin d + 19 a + 29 a + 48 c + 95 c 4-Methoxyglucobrassicin a a a = 221 ; b = 222 ; c = 211 ; d = 223. * Calculation is made by conversion of mol to g on dry weight basis. Goitrin, a naturally occurring compound in cruciferous vegetables, can easily be nitrosated if in contact with nitrites in gastrointestinal conditions, yielding the mutagenic compound -nitrosooxazolidone, with loss of sulphur. 220 Additionally, goitrin which is a decomposition product of progoitrin (Figure 4) is known to be strongly goitrogenic, inhibiting the synthesis of thyroid hormones, thyroxine, and tri-iodine-thyronine by a selective binding of iodine which prevents iodine intake by the thyroid gland

19 Health-afecting compounds in Brassicaceae The other decomposition products of glucosinolates, as mentioned before, are thiocyanates, isothiocyanates and oxazolidine-2- thiones (Figure 5), 50, 224 and have also been shown to be goitrogenic. The benzyl-, phenethyl-, allyl-isothiocyanate, and sulforaphane are formed through the hydrolysis of their naturally occurring precursor glucosinolates, glucotropaelin, gluconasturtiin, sinigrin and glucoraphanin, respectively, by myrosinase. 225 HO H 2 C HO C HO - OO 3 Progoitrin OH OH Myrosinase H O C H Goitrin CH 3 Figure 4 Conversion of progoitrin to goitrin by myrosinase However, under certain conditions, the glucosinolate aglycones may yield a nitrile rather than an isothiocyanate. itriles such as -lcyano-2-hydroxy-3-butene and 1-cyano-2-hydroxy-3,4-epithiobutane, are the most toxic of the normal glucosinolate hydrolysis products, with a human lethal dose of 170 and 178 mg/kg, respectively. 218 These negative effects of glucosinolates have led to research directed at finding methods to reduce the glucosinolate content in the seeds of some Brassica crops. 47 Other processes intended to avoid toxicity of the meal include heat treatment of the seeds prior to removal of the oil. This inactivates myrosinase and subsequent breakdown of glucosinolates when the meal is consumed. 218 High or low glucosinolate contents of the seed of some varieties of B. napus correlate positively with glucosinolate levels in the roots, at least during the early stages of in vitro plant development. 43 Glucosinolates are also responsible for the bitter acidic flavours of Brassicacea species 76 and the hydrolysis by-products of glucosinolates mentioned above, such as isothiocyanates, nitriles, and thiocyanates, are responsible for the hot and pungent taste of the mustard that is often objected to by consumers. 207 Many of these degradation products are volatile 226 and also play an important role in the characteristic aroma or off-odour of Brassicacae. 84 A great deal of research has been carried out on the volatiles of these species. 31

20 Chapter 2 Cruciferous vegetables, for example, have been reported to contain substantial quantities of isothiocyanates. 227 Volatiles and semivolatiles from B. oleracea L. var. botrytis seeds were identified as cyanides such as 4-(methylthio) butyl-cyanide, 3-(methylthio) propyl cyanide, and isothiocyanates such as 4-(methylthio) butylisothiocyanate. 228 In B. rapa var. perviridis, 6 isothiocyanates were detected in the steam volatiles and identified as sec-butylisothiocyanate, 3-butenylisothiocyanate, 4-pentenylisothiocyanate, benzylisothiocyanate, 2-phenylethylisothiocyanate, and 5- methylthiopentylisothiocyanate. Three nitriles were also detected and identified as 2-methyl-5-hexenenitrile, 3-phenylpropiononitrile, and 6- methylthiohexanonitrile. 84 In Brassica oleracea var. Botrytis 35 volatile and semi-volatile constituents were detected. 228 Dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, hexanal, 3-cis-hexen-1-ol, nonanal, ethanol, 228, 229 and hex-3(z)-enol were identified as major constituents representing, respectively, 30.2, 24.2, and 21.7% of the volatiles. 228 Various interesting bioactivities have also been 43, 230 reported for hydrolysis and breakdown products of glucosinolates, 218, 231, 232 such as strong bactericidal, antifungal properties, and health promoting effects for plants and humans. 47, 224 ome of these glucosinolates have a potential application in the industry, for example, an aqueous extract of B. nigra seeds might be included in industrial biofilms as an antimicrobial agent. 233 The breakdown products of glucosinolates assist in the activity of important naturally occurring, direct- acting antioxidants such as tocopherols and also enhance the synthesis of glutathione, one of the most abundant intracellular direct antioxidants. 234 Working on rapeseed oil cake (Brassica campestris L. subsp. napus), different antioxidant compounds (indolacetonitrile, -1- methoxy-1-(3,5-dimethoxy-4-hydroxyphenyl) ethane, 4-hydroxy-indolacetonitrile, and 4-hydroxy-phenyl-acetonitrile) were isolated, which showed a strong antioxidant activity as evaluated by the ferric thiocyanate method. 235 Certain glucosinolates, particularly the isothiocyanates and nitriles, have been shown to modify both xenobiotic metabolizing enzymes and induce cell cycle arrest and apoptosis. It is likely that a combination of these responses explains the chemo-preventive characteristics of Brassica and that a combination of different cruciferous vegetables could provide optimal protection. 225, 236 The isothiocyanate chemopreventive activity could be due to its powerful inhibition of different enzymes such as glutathione -transferases (GTs) in 32

21 Health-afecting compounds in Brassicaceae 237, 238 humans. Another potential cancer-blocking action, which was described for both intact and thioglucoside glucohydrolase-treated glucosinolates, as assessed by induction of GTs activity, was found to be dependent on the nature of the side chain of the parent glucosinolate. 239 Another naturally occurring isothiocyanate, sulforaphane, that is present in Brassica vegetables has been shown to block the formation of tumours 240 and together with 7-methylsulfinyl-heptyl isothiocyanates in broccoli (B. oleracea var. italica) extract exhibited an inhibitory effect on cancer cell invasion and matrix metallo-proteinase-9 activity in human breast cancer cells 213 and lowers the probability of acquiring colon and rectal cancers. 241 It was also proved to inhibit Helicobacter pylori infection, blocking gastric tumour formation. This suggests that broccoli consumption could prevent chronic atrophic gastritis induced by H. pylori infection and, thus, this type of stomach cancer. 242 aturally, the wide range of glucosinolates content among different groups of B. oleracea would result in significant differences in their health-promoting properties. 211 HO HO Myrosinase OH O OH C R OO 3 - R C Unstable intermediate HO HO OH O OH Glucose OH HO 4 + R C H R C O Thiocyanates R C ulfur Isothiocyanates Oxazolidine-2-thiones Figure 5 Glucosinolate degradation R 33

22 Chapter 2 Indole-3-carbinol (I-3-C) is another glucosinolate breakdown product found in vegetables of the Brassica genus (cabbage, broccoli sprouts, Brussels sprouts, cauliflower, bok choy, and kale). ome research points to this compound as a promising anticancer agent against prostate cancer and reducing the incidence and multiplicity of mammary tumours. 243, 244 Coinciding with these studies, oral administration of I-3-C has been shown to have a possible beneficial effect on estrogen metabolism in humans and epidemiological studies support the claim that high intakes of I-3-C may have a broad chemo-preventive effect. 245 Conversely, [5,6,11,12,17,18]-hexahydrocyclonona-[1,2-b:4,5-b:7,8-b]- tri-indole (CTr), a major digestive product of indole-3-carbinol, has been proved to exhibit strong estrogenic activities increasing proliferation of estrogen-dependent breast tumour cells. Thus, the contribution of CTr to the cancer preventive or cancer-promoting effects of I-3-C remains to be established. 246 In plants, levels of secondary metabolites, such as glucosinolates are controlled by a number of factors. Although it is possible to increase levels of glucosinolates in plants by genetic manipulation, in order to enhance a particular pharmacological benefit, such a step would be premature and must await a more thorough understanding of the extremely complex interactions of these compounds and their metabolites Conclusion Brassica vegetables represent a major part of the human diet all over the world providing nutritionally significant constituents, such as phenolic compounds, vitamins, fibres, soluble sugars, minerals, fat, and carotenoids. Cruciferous vegetables are a source of some very promising chemopreventive dietary constituents, which may protect against free radical damage and LDL oxidation implicated in the pathogenesis of cardiovascular diseases, as well as DA damage and cancer. This might be useful information from the point of view of identifying appropriate raw materials, rich in these protective components, for the development of safe food products and additives with appropriate antioxidant properties. As mentioned above, Brassica plants are rich in many metals including calcium and iron-containing compounds. However, there is substantial variation both within and between subspecies, which suggest, a difference in potential health benefits depending on genotype, as well as on the growth conditions and environment. This review provides a 34

23 Health-afecting compounds in Brassicaceae massive body of evidence supporting the nutritional value of Brassica vegetables and should ultimately lead the population to better food choices. 11 Future Perspectives Many anti-cancer agents are of plant origin, but their actual function or the mechanism behind the role they play in the plant has not yet been fully elucidated. For example, plant-derived molecules with known roles in plant cell death may be novel candidates for use in clinical oncology. But a better understanding of the molecular and cellular mechanism of action of such compounds and their structureactivity relationships is necessary for the development of new derivatives of these molecules with more favourable chemopreventive activities. Different classes of anti-cancer compounds merit continued research at a basic and pharmacological level in order to yield novel chemotherapeutic agents. However, in order to correctly evaluate the effect of such compounds in food, it is necessary to bear in mind that some constituents such as phenolic acids, tannins, and other anti-nutritional compounds may form complexes with nutritionally important compounds, reducing their bioavailability and thus lowering the nutritional value of Brassica products. Additional studies are needed to determine the amount of isothiocyanates or their metabolites that reach target tissues, and the concentration needed to exert biological effects. 225 Further elucidation of the protective mechanisms of food and the identification of active constituents is needed. Enhancing the phytonutrient content of plant foods through selective breeding or genetic improvement is a powerful tool for dietary disease prevention. However, most, if not all, of these bioactive compounds confer a bitter, acid, or astringent taste to the food which is rejected by most consumers. Moreover, in the past, some of these compounds have even viewed as plant-based toxins and, as a result, the food industry routinely removes these compounds from plant foods through selective breeding and a variety of debittering processes. This poses a dilemma for the designers of functional foods because increasing the content of bitter phytonutrients for health may clash with consumer choices. tudies on phytonutrients and health, taking sensory factors and food preferences into account, constitute an important area of research. Another aspect of these valuable Brassica vegetables that deserves full attention is the edaphic conditions in which they are grown. 35

24 Chapter 2 These plants can be biofortified by growing them in a high mineralcontaining medium, attaining high levels of nutritionally important minerals that can be used to produce dietary supplements. But this advantage which is due to their metal tolerance (and allows their use for phytoremediation as previously explained) can be negative as observed in crops that are irrigated with polluting metals. The excessive heavy metals (macro or micro nutrients in excess) and plant and human pathogenic microbes concentrated in the soil from this water cause stress conditions for plants. Quality parameters of Brassicacea vegetables are very susceptible to great changes with these stress conditions which produce different effects on the levels of Brassica vegetables metabolites, affecting their flavour and leading to the changes in nutritional value. tudies are needed to clarify the route of exposure, mechanisms of sensitization, and clinical importance of these phenomena. Another question about cruciferous vegetables is their flavonoid content. Epidemiological data indicate that the present rate of consumption of these vegetables is beneficial. However, earlier studies also raised the question on the advantages of recommending an increased consumption of Brassica vegetables and/or phytochemical supplements. One of the reasons for this lies in the flavonoid content of these vegetables which, as explained above, is quite high in some of the species. Unfortunately, the potentially toxic effects of excessive flavonoid intake are still largely ignored. It is known that at high doses, flavonoids may act as mutagens, that is, pro-oxidants that generate free radicals, so that their adverse effects may well outweigh their beneficial ones. It is imperative that further research be conducted to learn more about the toxicological properties of flavonoids, apart from other putative health promoting compounds in Brassica vegetables, thus clarifying the balance of potential adverse and beneficial effects included in their mechanisms of action. 36